Nucleotide sequences specific to Francisella tularensis and methods for the detection of Francisella tularensis

ABSTRACT

Described herein is the identification of nucleotide sequences specific to  Francisella tularensis  that serves as a marker or signature for identification of this bacterium. In addition, forward and reverse primers and hybridization probes derived from these nucleotide sequences that are used in nucleotide detection methods to detect the presence of the bacterium are disclosed.

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/400,892, filed Aug. 1, 2002, and entitled, “DNA DiagnosticsFrancisella Tularensis Species,” which is incorporated herein by thisreference.

The United States Government has rights in this invention pursuant toContract No. W-7405-ENG-48 between the United States Department ofEnergy and the University of California for the operation of LawrenceLivermore National Laboratory.

SEQUENCE LISTING IN COMPUTER READABLE FORM

The sequence listing information recorded in computer readable form isidentical to the written on paper sequence listing.

BACKGROUND

Francisella tularensis is the species of bacteria known to cause what iscommonly known as Tuleremia, a serious and sometimes fatal disease.Since the attack on the World Trade Center in New York of Sep. 11, 2001,there has been a growing concern that terrorists or rogue governmentswill use the Francisella tularensis bacterium as a weapon of massdestruction and instrument of terror. Since the events of Sep. 11, 2001,the United States Government has been developing reliable methods andsystems to detect the Francisella tularensis bacterium so that immediateand effective counter measures can be undertaken. The existing methodsfor detecting the Francisella tularensis bacterium are consideredinadequate because of the higher than acceptable rate of false positiveand false negative results. False positive results lead to confusionregarding whether the Francisella tularensis bacterium is actuallypresent and whether protective measures should immediately beimplemented. Conversely, false negative results would allow theFrancisella tularensis bacterium to remain undetected with consequentadverse impacts. A more reliable method of detecting the Francisellatularensis bacterium would reduce the occurrence of false positive andfalse negative results and provide decision makers with greaterconfidence in implementing appropriate counter measures.

SUMMARY OF THE INVENTION

An aspect of the invention includes the nucleotide sequences that areidentified in SEQ ID NOs:4, 8, 12, 16, 20, 24, 28 and 32 that arespecific to Francisella tularensis.

Another aspect of the invention includes a Forward Primer, thenucleotide sequences that are identified in SEQ ID NOs:1, 5, 9, 13, 17,21, 25, and 29 and any primers that are derived from these nucleotidesequences.

A further aspect of the invention is a Reverse Primer, the nucleotidesequences that are identified in SEQ ID NOs:2, 6, 10, 14, 18, 22, 26 and30 and any primers that are derived from these nucleotide sequences.

A further aspect of the invention is includes a Hybridization Probe, thenucleotide sequences that are identified in SEQ ID NOs:3, 7, 11, 15, 19,23, 27 and 31 and any probes that are derived from these nucleotidesequences.

This invention also includes a method for the detection of Francisellatularensis using the bacterium specific nucleotide sequence comprising:providing a sample in an environment that is suitable for isolatinggenomic DNA for amplification using PCR and under conditions suitablefor hybridization with a least one group of nucleotides consisting orforward primer, a reverse primer and a hybridization probe and detectingthe existence of Fracisella Tularensis specific nucleotide sequences bya nucleotide detection method, such as PCR and flurogenic 5′ nucleasePCR assay, wherein the existence of the nucleotide sequence indicatesthe presence of Francisella tularensis in the sample. BRIEF DESCRIPTIONOF THE SEQUENCES SEQ ID NO: 1 Primer SEQ ID NO: 2 Primer SEQ ID NO: 3Probe SEQ ID NO: 4 Amplicon SEQ ID NO: 5 Primer SEQ ID NO: 6 Primer SEQID NO: 7 Probe SEQ ID NO: 8 Amplicon SEQ ID NO: 9 Primer SEQ ID NO: 10Primer SEQ ID NO: 11 Probe SEQ ID NO: 12 Amplicon SEQ ID NO: 13 PrimerSEQ ID NO: 14 Primer SEQ ID NO: 15 Probe SEQ ID NO: 16 Amplicon SEQ IDNO: 17 Primer SEQ ID NO: 18 Primer SEQ ID NO: 19 Probe SEQ ID NO: 20Amplicon SEQ ID NO: 21 Primer SEQ ID NO: 22 Primer SEQ ID NO: 23 ProbeSEQ ID NO: 24 Amplicon SEQ ID NO: 25 Primer SEQ ID NO: 26 Primer SEQ IDNO: 27 Probe SEQ ID NO: 28 Amplicon SEQ ID NO: 29 Primer SEQ ID NO: 30Primer SEQ ID NO: 31 Probe SEQ ID NO: 32 Amplicon

DETAILED DESCRIPTION

Disclosed herein are nucleotide sequences located on different loci ofthe Francisella tularensis bacterium genome, and primers and thehybridization probes used in detecting the specific nucleotidesequences. Also disclosed is a method for identifying Francisellatularensis by analyzing samples taken from monitoring devices, such asair monitors, for the nucleotide sequences that are specific toFrancisella tularensis. As the nucleotide sequences that have beenidentified are unique to the Francisella tularensis bacterium, using theprimers and hybridization probes to detect the presence of theFrancisella tularensis bacterium is far more reliable than existingmethods and partly reduces the occurrence of false positive and falsenegative results.

Francisella tularensis is the bacterium that causes Tularemia, a diseasethat can be fatal if not detected and treated with appropriateantibiotics. The symptoms of Tularemia “could include sudden fever,chills, headaches, muscle aches, joint pain, dry cough, progressiveweakness, and pneumonia”, this information can be found at the internetaddress www.bt.cdc.gov/documentsapp/FactSheet/Tularemia/about.asp. It ison the Center for Disease Control and Prevention (CDC) list of possiblebacteria that has potential as a biological warfare weapon. The CDC hasdeveloped a list of possible pathogens that may be used as weapons ofmass destruction. Francisella tularensis has been listed in Category Aof possible diseases and agents. Those diseases and agents in Category Aare considered a high risk to national security because they “can beeasily disseminated or transmitted from person to person; result in highmortality rates and have the potential for major public health impact;might cause public panic and social disruption; and require specialaction for public health preparedness”, this information can be found atthe internet address www.bt.cdc.gov/agent/agentlist-category.asp.

A key element in developing defenses against the use of Francisellatularensis is the ability to quickly and accurately detect the presenceof the bacterium. Early detection will allow for the implementation ofeffective counter measures. Additionally, early detection will allow forthe identification and treatment of those that may have been exposed tothe bacterium. Early detection and treatment is essential for thetreatment of Tularemia because although the disease may be fatal, it isusually treatable with antibiotics upon early detection.

Existing detection methods have resulted in a higher than acceptablerate of false positive and false negative results. Such results areinadequate and can create confusion regarding the appropriatecountermeasures, if any, that should be undertaken because it is unclearwhether the bacterium is present or not. If the bacterium is notpresent, undertaking counter measures may cause undue expense and createunwarranted concern among those that may incorrectly believe they havebeen exposed.

Although the genome for Francisella tularensis has already been mapped,this alone is not sufficient to develop a reliable and accuratedetection mechanism because the current methods use nucleotide sequencesthat may be common to many different bacteria. Thus, existing detectionmethods could not distinguish between various bacteria, which results inhigher than acceptable false positive detection rates. Similarly, someexisting detection methods result in false negative results because theyare not sensitive enough to detect the bacterium.

Using a nucleotide sequence that is specific to results in a morereliable detection method.

In order to detect any of the eight amplicons specific to Francisellatularensis, a series of forward and reverse primers and hybridizationprobes were developed for each of the eight amplicons.

A typical assay can determine the presence of SEQ ID Nos 4 and 8 usingthe sequence specific primers and the hybridization probes. If there isa positive result for the presence of Francisella tularensis then anassay is run to determine the presence of additional amplicon sequences,as a means to double check for the presence of Francisella tularensis.

Identifications of the Francisella tularensis specific nucleotidesequences allows the presence of the bacteria to be detected fromenvironmental samples using PCR assay analysis and detection. PCR is atechnique utilized to amplify genomic DNA. Typical PCR reactions includeappropriate PCR buffers, nuclease polymerase and one or moreoligonucleotide primers and hybridization probes. Various modificationsof PCR techniques are possible as detailed in Current Protocols inMolecular Biology ed. F. M. Ausubel, R. Brent, D. D. Moore, K. Struhle,Massachusetts General Hospital and Harvard Medical School (1987) whichis hereby incorporated by reference. The following US patents describePCR and are incorporated herein by reference: U.S. Pat. No. 4,683,195;U.S. Pat. No. 4,683,202; U.S. Pat. No. 4,800,159.

One method that may be used for real-time PCR amplification anddetection is TaqMan®. The principles involved in the conventionalTaqman® 5′ exonuclease assay are described in detail by Holland et alin, Detection of specific polymerase chain reaction product by utilizingthe 5′—3′ exonuclease activity of Thermus aquaticus polynucleotidepolymerase, Proc Natl Acad Sci USA 88 (16):7276-80, 1991, which isherein incorporated by reference. TaqMan® real time detection can alsobe used to simultaneously detect a plurality of nucleic acid targetswhen it is used with multiplex PCR, which enables simultaneous detectionof more than one target sequence, thus enhancing detection accuracy. Afew examples of typical PCR instruments include the ABI prism 7700, theCepheid Smart Cycler, and the Bio-Rad iCycler. In order to use a PCRassay method for detection of the Francisella tularensis bacterium, thesample must be prepared to extract all DNA that may be present. Thefollowing is a protocol for the preparation of samples taken fromambient air monitoring devices for nucleotide detection usingfluorogenic 5′ nuclease PCR assay.

Assay Protocol

Definitions:

DNA—deoxyribonucleic acid

EDTA—ethylenediaminetetraacetic acid

PCR—polymerase chain reaction

PCR water—autoclaved water, then filtered

CT—cycle threshold—the cycle in which the fluorescence signal crosses auser defined threshold

FAM—reporter dye

TAMRA—quencher dye

Sample Preparation:

Exposed environmental filters are suspended in Sodium phosphate/EDTA,Tween buffer and bead beaten. The supernatant is filtered and washed toyield the genomic DNA extract. The extract is then subjected toreal-time polymerase chain reaction (PCR) assay using afluorescent-labeled probe. This process monitors a PCR reaction and thequantity of double-stranded product that is produced

Materials Needed:

A series of forward and reverse primers, a hybridization probe andpolymerase reagents specific to the first amplicon to be detected. Aseries of forward and reverse primers, a hybridization probe andpolymerase reagents specific to the second amplicon to be detected.

Bead beater Kit with the following labeled components:

a. 3 capped tubes containing a filter and beads

b. 3 yellow ultra free MC Centrifugal Filter Units

c. 6 blue microcon YM-100 filter units

d. 12 collection tubes

e. 7 PCR reaction mix-includes primer/probe and Taq-labeledA,B,C,D,E,F,G

f. 48-25,μl Smart Cycle reaction tubes

g. Sodium phosphate buffer/EDTA Teen buffer

h. PCR water

i. Inhibitory control DNA

j. Extra unlabeled tubes

Materials not provided in the bead beater kit:

a. Cepheid Smart Cycle

b. Microcentrifuge

c. Microfuge for Cepheid tubes

Preparation of DNA Extract:

This part of the assay protocol is performed in segregated work areasand in a biosafety cabinet using BSL 2 practices.

-   -   1. Add 400,μl of Sodium phosphate/ED TA Teen buffer to each of        the capped tubes containing a filter and beads. Screw cap        tightly.    -   2. Insert tubes one at a time into the bead beater.    -   3. Bead beat the capped tube for 3 minutes and a speed of 5000        rpm.    -   4. Remove capped tube from bead beater and place the tube on ice        for a minimum of 2 minutes to cool.    -   5. Wash Steps.    -    Spin capped filter tubes for 10 seconds (pulse spin) in        microcentrifuge.    -    Transfer approximately 400,μl of the supernatant to the yellow        top filter collection tube.    -    Spin the yellow top filter tube with the supernatant @ 7000 rpm        for 3 minutes.    -    Transfer the filtered liquid to a blue microcon filter on        collection tube #1.    -    Spin @ 7000 rpm for 1 minute. Check fluid level in the blue        microcon filter. If it is above the white base, pulse spin for        about 10 seconds to bring the level at or a little below the        white area. It could take more than one pulse spin to bring the        level down. This is approximately 100-200 μl of liquid.    -    Transfer this liquid on the top of the filter to a second blue        microcon filter on a clean collection tube #2. Tilt the tube at        a 45° angle and take off the liquid-do NOT vacuum the filter.    -    Add 400 μl of PCR water to the second blue microcon filter with        the added liquid.    -    Spin @ 7000 rpm for 2 minutes. Do not be spin dry; there should        be about 50-100 μl of liquid on top of the filter. Pulse spin if        the level is too high    -    Using clean, metal forceps remove the blue microcon filter from        the collection tube #2 and place the blue microcon filter on a        clean collection tube #3. Discard collection tube #2.    -    Add 400 μl PCR water to the blue microcon filter on collection        tube #3.    -    Spin @ 7000 rpm for 2 minutes. Do not be spin dry; there should        be about 50-100μl of liquid on top of the filter. Pulse spin if        the level is too high.    -    Remove the blue microcon filter. Place the blue microcon filter        on collection tube #4. Discard collection tube #3    -    Add 400 μl PCR water to the blue microcon filter on collection        tube #4.    -    Spin for 1 minute @ 7000 rpm. Check fluid level in the blue        microcon filter. If it is above the white base, pulse spin for        about 10 seconds to bring the level at or a little below the        white area. This could take more than one pulse spin. If the        level of liquid is at or a little below the white base, this is        approximately 100-200 μl of DNA extract. If for some reason the        pulse spin has brought the level of DNA extract down too low add        200μl PCR water and bring the level carefully to the white base        level by pulse spinning for less time).    -    Transfer the liquid on the top of the blue microcon filter        (your DNA extract) to the eppendorf tube.    -    If you cannot perform the PCR assay immediately, keep extract        refrigerated.        PCR Assay:

1. Thaw on ice each set of primer/probe/Taq polymerase sets. Once thawedPCR assay must begin. Do NOT refreeze. Keep on ice while testing.

2. Add 20 μl of each of the PCR reaction mixes for each amplicon and oneinhibitory control to the appropriately labeled Cepheid reaction tubes;e.g. 1-1A is for amplicon 1, filter 1, a set of primers, probe, Taqpolymerase.

-   -   a. Add 5 μl of DNA extract to each of the tubes-rinse tip1-2        times in the mix and discard the tip.    -   b. Use a clean tip for each reaction tube.    -   c. Each tube should have a total of 25 μl .

3. Add 15 μl of PCR reaction inhibitory mix to appropriately labeledCepheid tubes; e.g INHIB.

a. Add 5 μl DNA extract to each tube-rinse tip 1-2 times in the mix anddiscard tip.

b. Add 5 μl of DNA inhibitory control to each tube-rinse tip 1-2 timesin the mix nd discard the tip.

a. Use a clean tip for each reaction tube.

b. Each tube should have a total of 25 μl .

4. Include as controls:

-   -   NTC (no template control) for each set of primers/probe, Taq.

5. Spin Cepheid tubes in Cepheid microfuge for about 4 seconds. Thismixes the PCR reaction mix and DNA into the optic diamond area. Check tosee that the optic area is filled. Run Cepheid Smart Cycler Record allCT values (including 0) on the result sheet for the appropriate organismand filter.

6. CT values from 34 to 35 indicate negative readings—no Francisella DNAdetected. CT values below 34 indicate positive readings—Francisella DNAdetected.

Table 1 shows the results of assay runs that were performed using theabove described protocol. An assay set containing the primers and probefor each amplicon sequence was added to a sample containing either theFrancisella tularensis bacteria or Francisella philomiragia. TheFrancisella philomiragia bacteria is similar in genetic composition toits cousin Francisella tularensis. Francisella philomiragia was,therefore, used as a control to demonstrate that the primers and probederived from the Francisella tularensis specific amplicon coulddistinguish between genetic near neighbors, thereby demonstrating thespecificity of the amplicon sequence. Three different strains of thephilomiragia bacteria were used, 25016, 25017 and 25018. The Francisellatularensis and the Francisella philomiragia DNA was obtained formAmerican Type Culture Collection. The assay sets containing the probesand primers were obtained from various vendors such as ABI andBiosearch.

All assays were run three times with the exception of Francisellaphilomiragia #25018 which was run twice. The results show that the assayruns having the Francisella tularensis DNA in the sample had positiveresults, CT values of less than 34. Those assay runs having Francisellaphilomiragia in the sample had negative results, CT values of 34 to 35.These results show that the primers and probe derived from theFrancisella tularensis specific amplicon can distinguish between neargenetic neighbors of the Francisella bacteria, thus demonstrating thatthe nucleotide sequences in the amplicons are specific to Francisellatularensis.

ft5698r2C106.tF: Seq. ID No. 35 35 35 35 35 35 35 35 21, 22, 23ft5698r2C127.tF: Seq. ID No. 35 35 35 35 35 35 35 35 25, 26, 27ft5698r2C86.tF: Seq. ID No. 35 35 35 35 35 35 35 35 29, 30, 31

The nucleotide sequences disclosed herein are specific to Francisellatularensis. Accordingly, although air monitors are an effective methodof obtaining samples for analyses, a wide variety of other media andmethods may be used to provide the samples for analysis for theFrancisella tularensis bacterium.

All numbers expressing quantities of ingredients, constituents, reactionconditions, and so forth used in the specification and claims are to beunderstood as being modified in all instances by the term “about”.Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the subject matter presented herein areapproximations, the numerical values set forth in the specific examplesare reported as precisely as possible. Any numerical value, however,inherently contain certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

1. A composition comprising an Amplicon, a single strand sequence ofnucleic acids specific to Francisella tularensis, selected from thegroup consisting of SEQ ID NO:4, 8, 12, 16, 20, 24, 28 and
 32. 2. Acomposition comprising a single strand sequence of nucleic acids that iscomplimentary to the sequence of nucleic acids recited in claim 1 or anyportion thereof.
 3. A composition comprising a single strand sequence ofnucleic acids selected from the group consisting of SEQ ID NOs:1, 2, 3,5, 6, 7, 9, 10, 11, 13, 14, 15, 17, 18, 19, 21, 22, 23, 25, 26, 27, 29,30 and
 31. 4. A method comprising: (i) providing a sample; (ii) forminga mixture by adding the sample to a solution containing at least oneseries of nucleotide sequences having a forward primer, a reverse primerand a hybridization probe selected from the group consisting of SEQ IDNOs:1, 2, 3; 5, 6, 7; 9, 10, 11; 13, 14, 15;17, 18, 19; 21, 22, 23; 25,26, 27;29, 30, 31; under conditions suitable for isolating genomic DNAfor amplification using PCR and under conditions suitable forhybridization with said at least one series of nucleotide sequences; and(iii)subjecting the mixture to PCR:
 5. The method of claim 4 whereinsaid PCR comprises standard PCR.
 6. The method of claim 5, wherein saidPCR comprises fluorogenic 5′ nuclease PCR assay.
 7. A method comprising(i) providing a sample; (ii) forming a mixture by adding the sample to asolution containing at least one series of nucleodtide sequences havinga forward primer, a reverse primer and a hybridization probe selectedfrom the group consisting of SEQ ID NOs:1, 2, 3; 5, 6, 7; 9, 10, 11; 13,14, 15;17, 18, 19; 21, 22, 23; 25, 26, 27;29, 30, 31; under conditionssuitable for isolating genomic DNA for amplification using PCR and underconditions suitable for hybridization with said at least one series ofnucleotide sequences; and (iii) detecting the presence of at least oneAmplicon sequence by flurogenic 5′ nuclease PCR assay, wherein thepresence of said one Amplicon sequence indicates the existence ofFrancisella tularensis in the sample.